Our long-range goal is to obtain crystal structures of different conformations of the lactose permease of Escherichia coli (LacY) in order to understand the mechanism of lactose/H+ symport at the atomic level. LacY is a paradigm for the Major Facilitator Superfamily, as well as membrane proteins in general. Our first X-ray crystal structure of a conformationally restricted mutant of LacY (C154G) represents a major breakthrough as the first structure of a cation-coupled symporter. In the past grant period, we accomplished another breakthrough by solving an x-ray structure of wild-type LacY to a resolution of 3.6 E, an accomplishment that took well over a decade and required development of a new, general approach-maintaining bound phospholipids. By this means, we also improved resolution of the C154G LacY structure to a resolution of ~2.9 E and showed that sugar binding is an induced-fit phenomenon. However, all structures display the same inward-facing conformation: pseudo-symmetrical N- and C- terminal 6 transmembrane 1-helix bundles, most of which are irregular, surrounding a large internal hydrophilic cavity open to the cytoplasmic side and tightly closed on the periplasmic side. The residues that play major roles in galactopyranoside recognition and H+ translocation are clustered near the apex of the cavity and inaccessible from the periplasmic side. A mechanism consistent with the structure and many biochemical/biophysical approaches is proposed, the heart of which is alternative accessibility of the sugar- and H+-binding sites to either side of the membrane. Despite a wealth of biochemical/biophysical data showing that transport involves opening and closing of inward- and outward-facing cavities, structures are needed in a different conformation(s) in order to obtain the mechanism at the atomic level. We have obtained diffracting crystals of likely candidates that are approaching a resolution suitable for atomic model building. The main aims of this proposal are (i) to obtain structures of conformations of LacY other than inward facing; (ii) to obtain a structure of LacY that diffracts to a resolution sufficient to visualize bound water, which may play a direct role in H+ translocation. We will combine mutagenesis and chemical modification to induce conformations different from the inward-facing conformation, which is favored by crystallization. The proposed structures will be invaluable for understanding the mechanism of cation-coupled membrane transporters, a class of proteins that plays essential roles in many cellular functions and has broad impact on biology and medicine. PUBLIC HEALTH RELEVANCE: Membrane proteins represent a very significant percentage of the genomes sequenced, and although they are involved in a multitude of essential cellular functions and are targets for the world's most widely prescribed drugs, their structures are grossly underrepresented. The lactose permease (LacY), which physiologically catalyzes the coupled translocation of lactose and a hydrogen atom across the membrane of the bacterium Escherichia coli, represents a well-known model for a huge family of related membrane transport proteins, many of which are clinically important. LacY has been used to develop numerous techniques for studying of this type of membrane transport proteins. In order to understand its mechanism of action, however, it is essential to obtain structures of LacY in more than the single form that we have obtained, which is the purpose of this proposal.